This invention relates to a vessel for mixing a cell lysate. The invention also relates to a method of using the vessel to mix a cell lysate in order to obtain high-purity products such as nucleic acids or proteins for use in a variety of applications.
The alkaline lysis of bacterial cells is a well-established laboratory technique for recovering plasmid DNA from host cells (Birnboim et al., Methods Enzymol. 100, 243-255, 1983). The method involves the addition of a detergent, usually sodium dodecyl sulphate (SDS) and sodium hydroxide to a bacterial cell suspension followed by the addition of a strong neutralising solution, usually potassium acetate or glacial acetic acid, after a short period of incubation. This results in a solution containing large aggregates, also referred to as flocs, comprising denatured chromosomal DNA, protein, cell wall and/or membrane material. The flocs are removed from the solution by coarse filtration or centrifugation, leaving a solution containing plasmid DNA that can be further purified by a variety of standard procedures.
The method is based firstly on the chemical breakage of cell walls and membranes, followed by the solubilization and denaturation of cellular components in an alkaline solution. The ability of plasmid DNA to renature upon neutralisation and chromosomal DNAs inability to do likewise enables the isolation of plasmid DNA.
The method of alkaline lysis and the properties of the solutions produced at various stages of the alkaline lysis method have previously only been of academic interest. However, with the advent of human gene therapy, utilizing plasmid DNA obtained from host cells such as E. coli, there is now a requirement for the production of large quantities of very high purity plasmid DNA. The requirement of such large-scale manufacture has increased the need for new techniques so that the scale and robustness of operations can be increased to the level and standard required.
Chromosomal DNA is found in both prokaryotes and eukaryotes and is known to possess viscoelastic properties, which have previously been utilised to determine the size of the DNA molecules (Chase, et al., Biophys. J. 28, 93-105, 1979). The viscoelastic properties have also been recognised when the process of alkaline lysis has been used to isolate plasmid DNA from bacteria (Nienow, AIChE Annual Meeting, November 1998, Florida USA). One feature of the process is the change in the rheological properties of the solution at various stages of the procedure. Studies of these rheological properties have shown that the initial cell suspension exhibits Newtonian characteristics. The characteristics of a Newtonian fluid can be summarised by three criteria (1) constant viscosity, and when a fluid flows under laminar conditions: (2) shear stress is directly proportional to shear rate, and (3) the only stress generated is shear stress (Barnes et al., Introduction to Rheology, 1989 and Ciccolini et al., Biotechnology and Bioengineering, 60, 768-770, 1998). Cell suspensions have been found to have a viscosity that is very nearly constant, with a value very similar to that of water i.e. less than 3 mPa (Ciccolini et al., Biotechnology and Bioengineering, 60, 768-770, 1998). When the alkaline detergent solution is added, the properties of the solution change dramatically to give a solution that has a shear-dependent, relatively low apparent viscosity with a maximum value of about 22 mPas at a shear rate of 350 sxe2x88x921 when a solution of about 120 g wet cell weight 1xe2x88x921 is lysed. Despite the solution""s low apparent viscosity, it is highly viscoelastic and gives a large Weissenburg effect. The Weissenburg effect is also known as xe2x80x9crod climbingxe2x80x9d and is defined as the effect created when a shaft or rod rotates in a viscoelastic solution (Barnes et al., Introduction to Rheology, 1989). A Newtonian fluid would be forced toward the outer sides of a vessel by inertia, whereas an elastic fluid climbs up the shaft as a direct consequence of the normal stress (a characteristic of a viscoelastic fluid) which acts like a hoop around the shaft. The viscoelastic properties of the lysate solution have been characterised using a rheometer operated under the oscillatory mode. The phase angle of the solution was found to be 58xc2x0, wherein 0xc2x0 represents a solid and 90xc2x0 represents an inelastic fluid. This demonstrates the elastic properties of the solution. The relatively low apparent viscosity in combination with viscoelasticity is very unusual as most viscoelastic fluids also exhibit high apparent viscosities. On neutralisation, the viscoelasticity of the solution breaks down so that it once again becomes of low viscosity, close to that of water, with no signs of viscoelasticity.
Whilst acting as a critical step in the recovery and purification of plasmid DNA, the lysis and neutralisation step have the potential for generating additional contaminants such as small fragments of chromosomal DNA and irreversibly denatured plasmid DNA.
The problems are, at least partially, pH and time-related, specifically with respect to the period for which the plasmid DNA can be incubated in the alkaline detergent solution. Extended exposure to the concentrated alkaline solution may result in the formation of denatured plasmid DNA (Birnboim and Doly, Nuc.Acids.Res., 7, 1513, 1979 and International Patent Application WO 97/29190). Denatured plasmid DNA cannot be readily purified from renatured plasmid DNA and its presence therefore leads to significant loss of functional plasmid yield. It is therefore important that mixing is sufficiently vigorous to ensure localised extremes of pH are avoided and that exposures to these conditions are minimised.
If mixing is too vigorous at any stage of the procedure, strands of chromosomal DNA are physically broken up. The chromosomal DNA may be fragmented to a size where the fragments may renature upon neutralisation and be carried forward into the recovery and purification procedure. The fragmented chromosomal DNA will contaminate the solution and create a significant purification problem as it has similar properties to plasmid DNA and cannot be readily separated by techniques such as chromatography to the level required by regulatory authorities responsible for clinical products. Also, if the mixing is vigorous during neutralisation, the flocs formed become too small, due to mechanical stress and cannot be readily removed by centrifugation or filtration.
Clearly, any mixing process must be able to achieve rapid mixing without creating unwanted chemical or mechanical damage to the plasmid DNA, in addition limiting the mechanical damage to the chromosomal DNA and ideally without creating mechanical damage of the flocculated material. Similar problems occur when isolating proteins form cell cultures.
During the process, significant volume changes occur. The alkaline detergent added to the cell suspension is usually equal to or double the volume of the initial suspension and the neutralisation solution is usually the same volume as that of the initial (cell suspension. Consequently, the initial cell suspension may only represent 25% of the final volume to be mixed.
Any mixing must take account of these changes in volume so that efficient mixing can be carried out over a wide range of volumes. In addition to this, and especially in systems where more than one impeller is in operation, steps are preferably taken in order to minimise air entrapment in the solution by impellers intersecting the air/liquid interface. Such entrapment may cause mechanical damage and fragmentation of the chromosomal DNA.
At the laboratory scale, the process of alkaline lysis is achieved by xe2x80x9cgentle mixingxe2x80x9d such as the inversion of a test tube or bottle. This technique is clearly limited in scale and subject to operator influence. However, some companies do carry out this operation using volumes of up to 5 l (Oral Presentation by Schleef M., Bio-Europe 1997, Cambridge, England, 1997).
If plasmid DNA or other desired cell derived products are to be generated in a pure form suitable for clinical trials, a scaleable method of carrying out mixing during the lysis procedure is required to overcome the problems detailed above.
Methods using static mixers have been described in, for example, International patent application WO 97/23601 but do not address the problems of plasmid quality, yield and purity.
General discussions of methods for mixing solutions in vessels during the lysis process have been made by Marquet et al., (Bio Pharm, September, 26-37, 1995), but do not provide any description or data of a system that addresses the problems detailed above.
The present invention provides a vessel and methods for generating plasmid DNA and other desired cell-derived products in scales suitable for, for example, clinical trials. The vessel and methods avoid chemical and mechanical damage to plasmid DNA (or the desired product) and mechanical damage to chromosomal DNA during mixing during the lysis procedure such that high-purity plasmid DNA or other desired products can be obtained.
In one aspect, the present invention relates to a vessel for mixing a cell suspension or cell lysate.
In another aspect, the present invention relates to a method for using the vessel disclosed above in the lysis of cells of a suspension wherein efficient mixing is obtained without generating mechanical stresses sufficient to lead to substantial fragmentation of chromosomal DNA or flocs of aggregated material.
In yet another aspect, the present invention relates to a method for monitoring the degree of lysis or neutralisation in a cell suspension comprising measuring the torque on the axle of the impeller.
In a further aspect, the present invention relates to a method for preparing plasmid DNA and other desired products by monitoring the degree of lysis or neutralisation.
In yet a further a aspect, the present invention relates to plasmids and proteins prepared using the vessels and methods disclosed herein.
In still a further aspect, the present invention relates to a sectioned helical ribboned impeller.